Introduction and objectives Extracellular vesicles (EVs) are nanosized membrane-encapsulated particles produced by almost all living cells and loaded with various molecules (eg, lipids, nucleic acids, proteins). They play important roles in cell-to-cell communication by transporting and transferring their cargo to recipient cells. EVs may have distinct activities, depending on the producing cell, their functional cargo and their mode of action in recipient cells. EVs produced by bacterial pathogens contribute to pathogenicity as mediators of host-pathogen interactions. However, how pathogen derived-EVs act on host cells is still poorly documented. Here, we characterized EVs produced by the methicillin-resistant Staphylococcus aureus strain N315, and evaluated their impact on the expression of several inflammatory genes, as well as their routes of entry into human non-phagocytic cells. Materials and methods To mimic infection, N315 strain was grown in RPMI + 10% LB medium and N315-derived EVs were purified by size exclusion chromatography from culture supernatants. EV size and concentration were determined by nanoparticle tracking analysis. Whole-cell and EV proteome was identified by LC-MS. EV uptake by the human osteoblast-like MG-63 non-phagocytic cell line was evaluated by confocal microscopy and flow cytometry in presence of various transport inhibitors. The impact of EVs on the expression of several MG-63 immune related genes was determined by RT-qPCR. Results, discussion and conclusion S. aureus N315 produced EVs of a typical spherical shape with an average size around 90 nm. Proteomic comparison of whole cells and its derived EVs revealed the selective packing mechanisms of proteins into N315 EVs, notably of lipoproteins. In addition, EVs contained lipoteichoic acid, peptidoglycan and ribonucleic acid. MG-63 cells can internalize EVs in a dose- and time-dependent manner and via mainly dynamin-mediated endocytosis. EVs induced the expression of numerous immune and signaling genes such as IL-6, IL-8, MCP-1, IL1B, TLR2, TLR4 and TLR7. The expression of some them drastically decreased when EV uptake was blocked, while others remained unchanged. Altogether, our results showed the ability of N315 EVs to trigger both extracellular and intracellular signaling and via different bacterial components, bringing new insights into staphylococcal pathogenesis.